Effect of Urocortin on human osteoclasts differentiation

Abstract: Introduction Osteoarthritis (OA) is a degenerative joint disease characterised by articular cartilage and subchondral bone degradation, which affects approximately eight million people in the UK only. There is currently no cure for OA with treatment focussed on the amelioration of symptoms and eventual joint replacement surgery in some cases. Studies in a mouse model however suggest that Urocortin, a CRH family peptide, may have a dual role in preventing both the death of chondrocytes and the degradation of bone by osteoclasts. The aim of this study is to investigate the role of Urocortin (Ucn) in human osteoclasts maturation by using human primary osteoclasts differentiated from peripheral blood. MethodsOsteoclasts were generated from human peripheral blood using buffy coats from an anonymous donor. Peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation, cultured for 5 days in presence of M-CSF to expand the precursor population and then for a further 7 days with M-CSF and RANKL to generate osteoclasts in presence or absence of Ucn (10-7M). Osteoclast differentiation was assessed by morphology, Tartrate-Resistant Acid Phosphatase (TRAP) staining and RT-PCR for the expression of osteoclast differentiation markers (TRAP, Cathepsin K, Calcitonin Receptor, Intergrin beta 3), normalized against GAPDH expression. Cell Viability was assessed by Neutral Red and MTT assays . Results The presence of Ucn (10-7M) during differentiation, did not result in a significant decrease in cell viability as assessed by Neutral Red and MTT assays (n=4) but did result in inhibition of osteoclast differentiation as assessed by several different measures. Morphological assessment and TRAP analysis revealed that Ucn treated cells look smaller and rounder with less nuclei and decreased TRAP staining when compared to the larger, poly-nucleated, strongly TRAP positive cells in control samples. Semi-quantitative RT-PCR analysis confirms inhibition of TRAP expression along with decreased expression of other differentiation markers. ConclusionThese results indicate that Urocortin prevents human osteoclast differentiation and may therefore have the potential to reduce osteoclast resorption of subchondral bone. This, along with the previously reported chondroprotective properties of this peptide, may present an opportunity for future therapeutic intervention in OA